Chapter 4 Mechanisms of Anticancer Drugs

4 Mechanisms of anticancer drugs SARAH PAYNE AND DAVID MILES

Introduction Principles of chemotherapy Principles of tumour biology Classification of chemotherapeutic agents Limitations of cytotoxic agents Chemotherapy in head and neck cancer Choi Ch oicce of che hem mot oth her eraapy in hea eadd an andd nec eckk can ance cerr Chemotherapy strategies

34 34 34 37 40 40 40 40

Novel therapies for the future Other novel treatments Conclusion Key points Deficiencies in current knowledge and areas for future research References Further reading

41 44 44 45 45 45 46

SEARCH STRATEGY  The data in this chapter are supported by a Medline search using the key words chemotherapy and head and neck  neoplasms, and focus on mechanisms of action of current and experimental drugs.

INTRODUCTION

PRINCIPLES OF CHEMOTHERAPY 

The discovery of the toxic action of nitrogen mustards on cells of the haematopoietic system more than 50 years ago init in itial ially ly tri trigg gger ered ed re rese sear arch ch in into to th thee de deve velo lopm pmen entt of  cytoto cyt otoxic xic age agents nts.. The ini initia tiall pro promis misee of the these se dru drugs gs in thee ma th mana nage geme ment nt of ha haem emato atolo logi gica call an and d ot othe herr ra rare re malig ma lignan nancie ciess has not bee been n su susta stained ined and cur curee of the more common epithelial malignancies when metastatic, remains an elusive goal. Manyy of the cur Man curren rentt che chemot mother herape apeuti uticc age agents nts hav havee been bee n dis disco cover vered ed as a res result ult of scr screen eening ing com compou pounds nds vitro ro   against for cytoto cytotoxic xic poten potency  cy   in vit against muri murine ne and/ and/or or vivoo   against human hum an can cancer cer cel cells ls or   in viv against roden rodentt tumou tumourr models mod els.. Wi With th our bet better ter un under dersta standi nding ng of the mol moleecula cu larr ba basi siss of ca canc ncer er th ther eree is no now w in inter teres estt in tar targe gettdire di recte cted d dr drug ug th ther erap apie ies. s. Th Thee ai aim m be bein ingg to de deve velo lop p agen ag ents ts th that at ca can n mo modu dula late te or in inhi hibi bitt sp spec ecifi ificc mo mole le-cular cul ar tar targets gets ide identi ntified fied as bei being ng ess essent ential ial for tum tumour our growth.

Many forms of chemotherapy are targeted at the process of  cell division. The rationale being that cancer cells are more likely lik ely to be rep replica licatin tingg than nor normal mal cel cells. ls. Un Unfort fortuna unately  tely  as th their eir ac actio tion n is no nott sp speci ecific fic,, th they ey ar aree as asso socia ciate ted d wit with h significant toxicity. An understanding of the principles of  tumour biology and cellular kinetics is helpful to appreciate the mechanisms of action of cancer chemotherapy.

PRINCIPLES OF TUMOUR BIOLOGY  Cellular kinetics CELL CYCLE

Uncontrolled cell division is a result of interference in the normal balance of the cell cycle. The cell cycle is divided into a number of phases governed by an elaborate set of 

Chapter 4   Mechanisms of anticancer drugs ]  35

molecular switches (Figure 4.1). Normal nondividing cells are in G0. When actively recruited into the cell cycle they then pass through four phases: 1. G1: the growth phase in which the cell increases in size and prepares to copy its DNA; 2.   S (synthesis): which allows doubling of the chromosomal material;

3. G2: a further growth phase before cell division; 4.   M (mitosis): where the chromosomes separate and the cell divides. At the end of a cycle the daughter cells can either continue through the cycle, leave and enter the resting phase (G0) or become terminally differentiated.

MITOSIS

DNA STRUCTURE

Prophase: Chromatin condenses into chromosomes DNA is coiled into a helix.  This is wound round histone proteins and ultimately coiled to form chromosomes

Metaphase: Spindle forms from microtubules and chromosomes align at the equatorial plane

 Topoisomerase uncoils DNA 

Pentose sugar Phosphate group

 Anaphase: Sister chromatids separate

Telophase: Cell division

Nitrogenous base Mitosis

Disulphide bond Purine bases:  Adenine Guanine Pyrimidine bases: Cytosine  Thymine (DNA only) Uracil (RNA only)

G2: Cell prepares to divide

G1: Cell enlarges and makes new proteins

DNA REPLICATION

RNA AND PROTEIN PRODUCTION S-phase: DNA synthesis

Non template strand

DNA  DNA is unwound by DNA helicase and topoisomerases

Exon

Intron Exon Intron  Transcription

Nucleotides align and DNA polymerase catalyses strand elongation

RNA 

 Template strand

RNA processing

Introns spliced out leaving mRNA  DNA ligase joins the fragments together resulting in 2 new strands of DNA 

 Amino acid chain to form polypeptide mRNA  Ribosome

Figure 4.1

Cell cycle: possible targets for chemotherapy.

36 ]  PART 1  CELL BIOLOGY  TUMOUR GROWTH

The kinetics of any population of tumour cells is regulated by the following:   doubling time: the cell cycle time, which varies considerably between tissue types;    growth fraction : the percentage of cells passing through the cell cycle at a given point in time which is greatest in the early stages;    cell loss : which can result from unsuccessful division, death, desquamation, metastasis and migration. 

Tumours characteristically follow a sigmoid-shaped growth curve, in which tumour doubling size varies with tumour size. Tumours grow most rapidly at small volumes. As they  become larger, growth is influenced by the rate of cell death and the availability of blood supply.

Cell signalling Cells respond to their environment via external signals called growth factors. These interact with cell surface receptors that activate an internal signalling cascade. This ultimately acts at the DNA level through transcription factors that bind to the promoter regions of relevant genes, stimulating the cell cycle and influencing many  important processes including cell division, migration, programmed cell death (apoptosis). ONCOGENES

Protooncogenes are involved in controlling normal cell growth. Mutated forms, known as oncogenes, can lead to inappropriate stimulation of the cell cycle and excessive cell growth. Alternatively, malignancy can also arise secondary  to abnormal activation of a normal gene. The consequences of gene activation associated with tumour growth include:   excess growth factor production;   alteration of growth factor receptor genes so that they  are permanently switched on;    alteration of the intracellular cascade stimulating proliferation. 

TUMOUR SUPPRESSOR GENES

These act as a natural brake on cell growth. Usually both alleles need to be lost for their function to be affected. This can have several important effects, which include:  impairment of the inhibitory signals influencing receptor genes or intracellular signalling;    loss of the counter signals controlling protooncogene function;   inhibition of apoptosis, often as a consequence of a mutation of p53, the protein associated with DNA repair. 

Metastatic spread A tumour is considered malignant when it has the capacity to spread beyond its original site and invade surrounding tissue. Normally cells are anchored to the extracellular matrix by cell adhesion molecules, including the integrins. Abnormalities of the factors maintaining tissue integrity will allow local invasion and ultimately  metastases of the tumour cells.

Mechanism of cell death There are two main types of cell death: apoptosis and necrosis. Necrotic cell death is caused by gross cell injury and results in the death of groups of cells within a tissue. Apoptosis is a regulated form of cell death that may be induced or is preprogrammed into the cell (e.g. during development) and is characterized by specific DNA changes and no accompanying inflammatory response. It can be triggered if mistakes in DNA replication are identified. Loss of this protective mechanism would allow mutant cells to continue to divide and grow, thereby conserving mutations in subsequent cell divisions. Many cytotoxic anticancer drugs and radiotherapy act by inducing mutations in cancer cells which are not sufficient to cause cell death, but which can be recognized by the cell, triggering apoptosis. FRACTIONAL CELL KILL HYPOTHESIS AND DRUG DOSING

Theoretically the administration of successive doses of chemotherapy will result in a fixed reduction in the number of cancer cells with each cycle.1 A gap between cycles is necessary to allow normal tissue recover. Unfortunately, these first-order dynamics are not observed in clinical practice. Factors such as variation in tumour sensitivity and effective drug delivery with each course result in an unpredictable cell response. Clinical responses to antitumour therapies are defined by arbitrary criteria that have been used as part of the evaluation process in assessing the potential utility of  novel agents.  Tumour size: –  complete response is defined as the apparent disappearance of the tumour;  partial response represents a reduction of more – than 50 percent; –  progression is defined as an increase in tumour size by more than 25 percent;  stable disease is an intermediate between partial – response and tumour progression.    Tumour products: –  biochemical or other tests can be used to assess response, including circulating tumour markers. 

Chapter 4   Mechanisms of anticancer drugs ]  37

CLASSIFICATION OF CHEMOTHERAPEUTIC AGENTS Classification according to phase-specific toxicity Cytotoxic drugs can be classified according to whether they are more likely to target cells in a particular phase of  their growth cycle. More crudely, they can also be divided into whether they are more toxic to cells that are actively  dividing rather than cells in both the proliferating and resting phases. PHASE-SPECIFIC CHEMOTHERAPY 

These drugs, such as methotrexate and vinca alkaloids, kill proliferating cells only during a specific part or parts of the cell cycle. Antimetabolites, such as methotrexate, are more active against S-phase cells (inhibiting DNA synthesis) whereas vinca alkaloids are more M-phase specific (inhibiting spindle formation and alignment of  chromosomes). Attempts have been made to time drug administration in such a way that the cells are synchronized into a phase of the cell cycle that renders them especially sensitive to the cytotoxic agent. For example, vinblastine can arrest cells in mitosis. These synchronized cells enter the S-phase together and can be killed by a phase-specific agent, such as cytosine arabinoside. Most current drug schedules, however, have not been devised on the basis of cell kinetics.

ALKYLATING AGENTS

These highly reactive compounds produce their effects by  covalently linking an alkyl group (R-CH2) to a chemical species in nucleic acids or proteins. The site at which the cross-links are formed and the number of cross-links formed is drug specific. Most alkylating agents are bipolar, i.e. they contain two groups capable of reacting with DNA. They can thus form bridges between a single strand or two separate strands of DNA, interfering with the action of the enzymes involved in DNA replication. The cell then either dies or is physically unable to divide or triggers apoptosis. The damage is most serious during the S-phase, as the cell has less time to remove the damaged fragments. Examples include:   nitrogen mustards (e.g. melphalan and chlorambucil);   oxazaphosphorenes (e.g. cyclophosphamide, ifosfamide);   alkyl alkane sulphonates (busulphan);    nitrosureas (e.g. carmustine (BCNU), lomustine (CCNU));    tetrazines (e.g. dacarbazine, mitozolomide and temozolomide);    aziridines (thiopeta, mitomycin C);    procarbazine. 

HEAVY METALS Platinum agents

These drugs, for example alkylating agents and platinum derivatives, have an equal effect on tumour and normal cells whether they are in the proliferating or resting phase. They have a linear dose–response curve; that is, the greater the dose of the drug, the greater the fractional cell kill.

These include carboplatin, cisplatin and oxaliplatin. Cisplatin is an organic heavy metal complex. Chloride ions are lost from the molecule after it diffuses into a cell allowing the compound to cross-link with the DNA strands, mostly to guanine groups. This causes intra- and interstrand DNA cross-links, resulting in inhibition of  DNA, RNA and protein synthesis. Carboplatin has the same platinum moiety as cisplatin, but is bonded to an organic carboxylate group. This leads to increased water solubility and slower hydrolysis that has an influence on its toxicity profile. It is less nephrotoxic and neurotoxic, but causes more marked myelosuppression. Oxaliplatin belongs to a new class of platinum agent. It contains a platinum atom complexed with oxalate and a bulky diaminocyclohexane (DACH) group. It forms reactive platinum complexes that are believed to inhibit DNA synthesis by forming interstrand and intrastrand cross-linking of DNA molecules. Oxaliplatin is not generally cross-resistant to cisplatin or carboplatin, possibly due to the DACH group.

Classification according to mechanism

ANTIMETABOLITES

Classifying cytotoxic drugs according to their mechanism of action is the preferred system in use between clinicians.

Antimetabolites are compounds that bear a structural similarity to naturally occurring substances such as vitamins, nucleosides or amino acids. They compete with

CELL CYCLE-SPECIFIC CHEMOTHERAPY 

Most chemotherapy agents are cell cycle-specific, meaning that they act predominantly on cells that are actively  dividing. They have a dose-related plateau in their cell killing ability because only a subset of proliferating cells remain fully sensitive to drug-induced cytotoxicity at any  one time. The way to increase cell kill is therefore to increase the duration of exposure rather than increasing the drug dose. CELL CYCLE-NONSPECIFIC CHEMOTHERAPY 

38 ]  PART 1  CELL BIOLOGY  the natural substrate for the active site on an essential enzyme or receptor. Some are incorporated directly into DNA or RNA. Most are phase-specific, acting during the S-phase of the cell cycle. Their efficacy is usually greater over a prolonged period of time, so they are usually given continuously. There are three main classes.

sulphur group replaces the keto group on carbon-6 in these compounds. In many cases, the drugs require initial activation. They are then able to inhibit nucleotide biosynthesis by direct incorporation into DNA.

CYTOTOXIC ANTIBIOTICS

Folic acid antagonists

Most antitumour antibiotics have been produced from bacterial and fungal cultures (often  Streptomyces  species). They affect the function and synthesis of nucleic acids in different ways.

Methotrexate competitively inhibits dihydrofolate reductase, which is responsible for the formation of tetrahydrofolate from dihydrofolate. This is essential for the generation of a variety of coenzymes that are involved in the synthesis of  purines, thymidylate, methionine and glycine. A critical influence on cell division also appears to be inhibition of the production of thymidine monophosphate, which is essential for DNA and RNA synthesis. The block in activity of  dihydrofolate reductase can be bypassed by supplying an intermediary metabolite, most commonly folinic acid. This is converted to tetrahydrofolate that is required for thymidylate synthetase function ( Figure 4.2).

  Anthracyclines  (e.g. doxorubicin, daunorubicin, epirubicin) intercalate with DNA and affect the topoiosmerase II enzyme. This DNA gyrase splits the DNA helix and reconnects it to overcome the torsional forces that would interfere with replication. The anthracyclines stabilize the DNA tomoisomerase II complex and thus prevent reconnection of the strands.    Actinomycin D   intercalates between guanine and cytosine base pairs. This interferes with the transcription of DNA at high doses. At low doses DNA-directed RNA synthesis is blocked.    Bleomycin  consists of a mixture of glycopeptides that cause DNA fragmentation.    Mitomycin C  inhibits DNA synthesis by cross-linking DNA, acting like an alkylating agent. 

Pyrimidine analogues

These drugs resemble pyrimidine molecules and work by  either inhibiting the synthesis of nucleic acids (e.g. fluorouracil (Figure 4.3)), inhibiting enzymes involved in DNA synthesis (e.g. cytarabine, which inhibits DNA polymerase) or by becoming incorporated into DNA (e.g. gemcitabine), interfering with DNA synthesis and resulting in cell death.

SPINDLE POISONS

Purine analogues

 Vinca alkaloids

These are analogues of the natural purine bases and nucleotides. 6-Mercaptopurine (6MP) and thioguanine are derivatives of adenine and guanine, respectively. A

The two prominent agents in this group are vincristine and vinblastine that are extracted from the periwinkle

Dihydrofolate reductase Methotrexate blocks here

Dihydrofolate

Tetrahydrofolate Folinic acid bypasses the block here

Thymidine monophosphate

RNA, DNA production

Deoxyuridine monophosphate

Thymidylate synthase works here

Methotrexate also blocks here

Figure 4.2 Mechanism of action of cytotoxic drugs: methotrexate.

Chapter 4   Mechanisms of anticancer drugs ]  39

U

UMP

PRPP

PP

5-FU

dUMP

NADP+

NADPH

 

5-FUMP

5-FdUMP

(a) O HN O HO

P

O O

C

N O

Thymidylate synthase

O

5-FdUMP

O−

CH3

HN

dUMP

dTMP O

OH OH Methylene-H4-folate

HO H2-folate

(b)

P

O O

C

N O

O−

OH OH

Figure 4.3 Mechanism of action of cytotoxic drugs: Fluorouracil. 5-Fluorouracil (5FU) can participate in many reactions in which uracil would normally be involved. Firstly, it has to be converted to its active form, 5-fluoro-2 deoxyuridine monophophate (5-FdUMP) (a). This then interferes with DNA synthesis by binding to the enzyme thymidylate synthetase, causing it to be inactivated  (b). The binding can be stabilized by the addition of folinic acid. 5FdUMP, 5-fluorodeoxyuridine monophosphate; 5FU, 5-fluorouracil; 5FUMP, 5-fluorouridine monophosphate; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophophase; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; PP, pyrophosphate; PRPP, 5-phospho-alpha-D-ribose 1-diphosphate; U, uracil; UMP, uridine monophosphate.

plant. They are mitotic spindle poisons that act by binding to tubulin, the building block of the microtubules. This inhibits further assembly of the spindle during metaphase, thus inhibiting mitosis. Although microtubules are important in other cell functions (hormone secretion, axonal transport and cell motility), it is likely that the influence of this group of drugs on DNA repair contributes most significantly to their toxicity. Other newer examples include vindesine and vinorelbine. Taxoids

Paclitaxel (Taxol) is a drug derived from the bark of the pacific yew,   Taxus brevifolia. It promotes assembly of  microtubules and inhibits their disassembly. Direct activation of apoptotic pathways has also been suggested to be critical to the cytotoxicity of this drug.2 Docetaxel (Taxotere) is a semisynthetic derivative.

reaction. They are involved in DNA replication, chromatid segregation and transcription. It has previously been considered that the efficacy of topoisomerase inhibitors in the treatment of cancer was based solely on their ability to inhibit DNA replication. It has now been suggested that drug efficacy may also depend on the simultaneous manipulation of other cellular pathways within tumour cells.3 The drugs are phase-specific and prevent cells from entering mitosis from G2. There are two broad classes: Topoisomerase I inhibitors

Camptothecin, derived from   Camptotheca acuminata (a Chinese tree), binds to the enzyme–DNA complex, stabilizing it and preventing DNA replication. Irinotecan and topetecan have been derived from this prototype. Topoisomerase II inhibitors

TOPOISOMERASE INHIBITORS

Topoisomerases are responsible for altering the 3D structure of DNA by a cleaving/unwinding/rejoining

Epipodophyllotoxin derivatives (e.g. etoposide, vespid) are semisynthetic derivatives of  Podophyllum peltatum , the American mandrake. They stabilize the complex between

40 ]  PART 1  CELL BIOLOGY  topoisomerase II and DNA that causes strand breaks and ultimately inhibits DNA replication.

CHEMOTHERAPY STRATEGIES Combination chemotherapy

LIMITATIONS OF CYTOTOXIC AGENTS There are a number of problems with the safety profile and efficacy of chemotherapeutic agents. Cytotoxics predominantly affect rapidly dividing cells so do not specifically target cancer cells in the resting phase. They  also only influence a cell’s ability to divide and have little effect on other aspects of tumour progression such as tissue invasion, metastases or progressive loss of differentiation. Finally, cytotoxics are associated with a high incidence of adverse effects. The most notable examples include bone marrow suppression, alopecia, mucositis, nausea and vomiting.

Combinations of cytotoxic agents are widely used for many cancers and may be more effective than single agents. Possible explanations for this include:  exposure to agents with different mechanisms of  action and nonoverlapping toxicities;   reduction in the development of drug resistance;   the ability to use combinations of drugs that may be synergistic. 

In practice, the predominant dose-limiting toxicity of  many cytotoxic drugs is myelosuppression and this limits the doses of individual drugs when used in combination.

CHEMOTHERAPY IN HEAD AND NECK CANCER

Adjuvant chemotherapy

Worldwide, squamous cell cancer of the head and neck accounts for an estimated 500,000 new cancer cases per year. One-third of these patients present with early  stage disease that is amenable to cure with surgery or radiotherapy alone. The remaining patients usually  present with locally advanced disease. Unfortunately, this group exhibit high recurrence rates of approximately  65 percent despite radical surgery and radiotherapy. To date, the addition of chemotherapy has not changed this. It has, however, allowed improved organ preservation when combined with radiotherapy and has led to a reduction in rates of distant metastases. Chemotherapy  also has a role in the palliative treatment of advanced disease. Currently, surgery or radiotherapy are the standard curative options for early stage head and neck cancer. Chemotherapy in combination with surgery, radiotherapy  or both is employed for locoregionally advanced disease. Stage IV disease is managed with palliative chemotherapy  (see also Chapter 200, Developments in radiotherapy for head and neck cancer).

This is the use of chemotherapy in patients known to be at risk of relapse by virtue of features determined at the time of definitive local treatment (e.g. tumour grade, lymph node status, etc.). The intention of adjuvant chemotherapy is therefore the eradication of micrometastatic disease. Randomized trials assessing the use of adjuvant chemotherapy for the patients with head and neck squamous carcinoma do not suggest a significant benefit.5 [****]

CHOICE OF CHEMOTHERAPY IN HEAD AND NECK CANCER The single agents active in head and neck cancer, with response rates between 15 and 40 percent, include methotrexate, cisplatin, carboplatin, fluorouracil, ifosfamide, bleomycin, paclitaxel and docetaxel. Cisplatin is particularly popular for use either as a single agent or in combination with other drugs because for a long time it was viewed as one of the most active drugs in squamous head and neck cancer.4 Taxoids and gemcitabine are now  gaining favour and are being incorporated into many  current drug trials. [***]

Neoadjuvant chemotherapy Neoadjuvant, or induction chemotherapy, is the use of  chemotherapy before definitive surgery or radiotherapy in patients with locally advanced disease. The intention of  this strategy is to improve local and distant control of the disease in order to achieve greater organ preservation and overall survival. Numerous phase III trials have considered the benefit of  neoadjuvant chemotherapy followed by definitive surgery, by surgery and radiotherapy, or by radiotherapy alone as compared to definitive management without chemotherapy. Unfortunately, these studies have not demonstrated a survival advantage. To date, only subset analyses of trials using neoadjuvant cisplatin and 5-fluorouracil combination chemotherapy compared with locoregional treatment alone have shown a small survival gain.5 In addition, neoadjuvant chemotherapy has been shown to have little impact on reducing locoregional failure. This is perhaps surprising given the consistently observed high initial tumour response rates of up to 70–85 percent. The role of neoadjuvant chemotherapy therefore continues to remain controversial and further studies are planned, particularly looking at more effective drug combinations. [****]

Chapter 4   Mechanisms of anticancer drugs ]  41

Concurrent chemoradiation This involves the synchronous use of chemotherapy and radiotherapy. Multiple randomized trials comparing concurrent radiotherapy and chemotherapy with radiotherapy  alone have shown significant improvement in locoregional control, relapse-free survival and overall survival rates in patients with locally advanced, unresectable disease.6 These results may reflect the influence of chemotherapy  on micrometastatic disease or its ability to enhance tumour radiosensitivity.7 Some chemotherapy agents are recognized to be more active in certain radioresistant cell types. Other drugs may act synergistically with radiotherapy by hindering the repair of radiation-induced DNA damage (cisplatin), by synchronizing or arresting cells during radiosensitive phases (hydroxyurea, paclitaxel) or by hindering regrowth between fractions of treatment. Many different drug combinations and radiation schedules have been evaluated. Each combination clearly  has unique toxicities, risks and benefits. At present, there is still debate regarding the optimum chemoradiotherapy  regimen that should become the standard of care. [****/**]

High-dose chemotherapy Many chemotherapy drugs have a linear dose–response curve, but their use at high doses is limited by  myelosuppression. This may be overcome by using bone marrow or peripheral stem cell infusions. While highdose chemotherapy appears to have a role in the management of leukaemias, myeloma and certain lymphomas, little benefit has been demonstrated in common solid tumours. [****]

Chemoprevention This is a novel approach with the aim of reversing or halting carcinogenesis with the use of pharmacologic or natural agents. Retinoids have been tested in head and neck carcinogenesis both in animal models and against oral premalignant lesions and in the prevention of secondary tumours in humans, with initial encouraging results.8, 9 Studies are also looking at the benefit of  using cyclo-oxygenase 2 (COX-2) inhibitors in a similar role.10 [**]

NOVEL THERAPIES FOR THE FUTURE Despite the introduction of new cytotoxic drugs, such as antimetabolites (capecitabine) and topoisomerase I inhibitors, the management of advanced head and neck  cancer remains challenging. Over the last years interest has focussed more on novel agents with a more targetted mechanisms of action.

Targeted therapy aims to specifically act on a welldefined target or biologic pathway that, when inactivated, causes regression or destruction of the malignant process. The main strategies of research have looked at the use of monoclonal antibodies or targeted small molecules.

Monoclonal antibodies In the early 1980s, it became apparent that targetted therapy using monoclonal antibodies (MAb) might be useful in the detection and treatment of cancer. Monoclonal antibodies can be derived from a variety of sources:   murine: mouse antibodies;   chimeric: part mouse/part human antibodies;   humanized: engineered to be mostly human;    human: fully human antibodies.  

Murine monoclonal antibodies may themselves induce an immune response that may limit repeated administration. Humanized and, to a lesser extent, chimeric antibodies are less immunogenic and can be given repeatedly. There are several proposed mechanisms of action of  monoclonal antibodies.11 These include:   direct effects: –   induction of apoptosis; –  inhibition of signalling through the receptors needed for cell proliferation/function;   anti-idiotype antibody formation, determinants – amplifying an immune response to the tumour cell;    indirect effects: –   antibody-dependent cellular cytotoxicity (ADCC, conjugating the ‘killer cell’ to the tumour cell); –   complement-mediated cellular cytotoxicity  (fixation of complement leading to cytotoxicity). 

A desirable target for MAbs would have the following properties:  wide distribution on tumour cells;   high level of expression;    bound to tumour, allowing cell lysis;    absent from normal tissues;   trigger activation of complement on MAb binding;    limited antigenic modulation of target. 

Antibodies have also been used as vectors for the delivery  of drugs and radiopharmaceuticals to a target of tumour cells. The earliest and most successful clinical use of antibodies in oncology has been for the treatment of haematological malignancies. Interest in the development of  antibodies for solid tumours has become increasingly  popular, especially with respect to the epidermal and vascular endothelial growth factor receptors.

42 ]  PART 1  CELL BIOLOGY 

Epidermal growth factor receptor biology

lipophilic region and an intracellular protein tyrosine kinase domain (Figure 4.4). When a substrate binds to the receptor, the ligand–receptor complex dimerizes and is internalized by the host cell. This activates an intracellular protein kinase by autophosphorylation, which in

Epidermal growth factor receptor (EGFR) biology is a 170-kDa transmembrane protein composed of an extracellular ligand-binding domain, a transmembrane

Growth factor

Growth factor

 Tyrosine kinase receptor

 Activation via phosphorylation (P)

P

Signal transducer

P

P P

Casade of protien phosphorylation

Secondary messenger

Signal transducer

Nuclear targets

c-fos

c-jun

c-myc

Effect of gene activation

Proliferation/  maturation

Chemotherapy/  radiotherapy resistance

Survival/  anti-apoptosis

 Angiogenesis

Metastasis

Figure 4.4 Simplified epidermal growth factor receptor signal transduction pathways and opportunities for intervention. GRB2, growth factor receptor binding protein 2; MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal related kinase; SOS, guanine nucleotide exchange factor (son of sevenless); c-fos, c-jun and c-myc, nuclear targets involved in gene transcription/cell cycle progression; P, phosphate; TGFa, transformation growth factor a; PI3-K, phosphotidyinositol 3; AKT, serine/threonine kinase, prosurvival protein; STAT, signal transducing activation of transcription.

Chapter 4   Mechanisms of anticancer drugs ]  43

turn activates signal transduction pathways, influencing cell function. This can lead to cell proliferation, as well as invasion and metastasis. Several investigators have described amplification of the EGFR gene and overexpression of the EGFR surface membrane protein in a large number of human cancers, including squamous cell carcinoma of the head and neck. 12 Overexpression is associated with increased proliferative capacity and metastatic potential and is an independent indicator of poor prognosis.13 Blockade of the EGFR  pathway has been shown to inhibit the proliferation of  malignant cells and also appears to influence angiogenesis, cell motility and invasion. 14 Various strategies have been investigated to manipulate EGFR.

Monoclonal antibodies against epidermal growth factor receptor MAb technology has been directed against EGFR. The chimeric IgG antibody cetuximab (C225) has the binding affinity equal to that of the natural ligand and can effectively block the effect of epidermal growth factor and transforming growth factor a.6 It has been shown to enhance the antitumour effects of chemotherapy and radiotherapy in preclinical models. 15, 16, 17 More recently, cetuximab has been evaluated alone and in combination with radiotherapy and various cytotoxic chemotherapeutic agents in a series of phase II and III studies involving patients with head and neck cancers.15, 18 The studies are encouraging, but it is still too early to determine the exact role the antibody will play in treatment regimens.

Targeted small molecules against epidermal growth factor receptor Gefitinib (Iressa) and erlotinib (Tarceva) are orally active epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) that block the EGFR signalling cascade, thereby inhibiting the growth, proliferation and survival of many solid tumours. They have single agent activity in patients with recurrent or metastatic head and neck  cancer, and have an acceptable safety profile compared with conventional chemotherapy.19, 20 Results of phase III trials are awaited and will help determine their optimal use in head and neck cancers. Interestingly, one of the noted side effects of the drugs is an acneiform rash. Analysis of phase II trials of  erlotinib in nonsmall-cell lung cancer, head and neck  cancer and ovarian cancer shows a significant association between the rash severity and objective tumour response and overall survival.21 Similar findings have been made with cetuximab and gefitinib. This association suggests that the rash may serve as a marker of response to treatment and could be used to guide treatment to obtain optimal dose.

Despite these successes, these agents have modest activity when used as single agents in unselected patients. It is clear that the clinical development of these agents is far from simple. It is important that we try to understand better the biological and clinical criteria for patient selection and also how best to use the different available agents. The recent discovery of EGFR mutations and the potential identification of other markers that might predict patient response could help to optimize the use of these agents in the future.

Inhibitors of angiogenesis Angiogenesis is the process of new blood vessel formation, triggered by hypoxia and regulated by numerous stimulators and inhibitors (Figure 4.5). It is vital for cancer development. A tumour cannot extend beyond 2–3 mm without inducing a vascular supply. New vessels develop on the edge of the tumour and then migrate into the tumour. This process relies on degradation of the extracellular matrix surrounding the tumour by  matrix metalloproteinases, such as collagenase, that are expressed at high levels in some tumour and stromal cells. Angiogenesis is then dependent on the migration and proliferation of endothelial cells. It has been found that antiangiogenic agents tend to be cytostatic rather than cytotoxic, hence stabilizing the tumour and preventing spread. As a consequence, they  may be valuable for use in combination with cytotoxic drugs, as maintenance therapy in early-stage cancers or as adjuvant treatment after definitive radiotherapy or surgery. There is evidence to support the fact that suppressing angiogenesis can maintain metastases in a state of dormancy.22 Interestingly, development of resistance does not appear to be a feature of these drugs.23 [**]

 Vascular endothelial growth factor receptor Vascular endothelial growth factor is a multifunctional cytokine released in response to hypoxia and is an important stimulator of angiogenesis. It binds to two structurally related trans-membrane receptors present on endothelial cells, called Flt-1 and KDR. High VEGF protein and receptor expression has been demonstrated in certain head and neck cancers and is associated with a higher tumour proliferation rate and worse survival.24

Monoclonal antibodies against vascular endothelial growth factor receptor Bevacizumab (Avastin) is a humanized murine monoclonal antibody targeting VEGF. It is the first antiangiogenic drug to have induced a survival advantage in cancer therapy, within a randomized trial of irinotecan,

44 ]  PART 1  CELL BIOLOGY 

Tumour Inflammatory cells

Proangiogenic factors produced by tumours and inflammatory cells Cause cell differentation, division and migration to form new blood vessels Blood vessel Pro-angiogenic factors

Anti-angiogenic factors

Fibroblast growth factor

 Thrombospondin I

Placental growth factor

 Angiostatin

 Vascular endothelial growth factor

Interferon alpha

 Transforming growth factors

Prolactin

 Angiogenin

Metallo-proteinase inhibitors

Interleukin-8

Endostatin

Hepatocyte growth factor

Platelet factor 4

Platelet derived enthothelial cell growth factor

Placental profliferin-related protein

5-fluorouracil, leucovorin combined with bevacizumab or placebo in metastatic colorectal cancer.25 The use of  bevacizumab in head and neck cancer is supported by  data from preclinical studies.26 Currently, clinical trials are exploring the feasibility and the therapeutic potential of a combination of bevacizumab and EGFR-targeted drugs.27

OTHER NOVEL TREATMENTS There are now a large number of new types of agents entering all phases of clinical trials. To date, they have met with variable success. It is important to mention a few  drugs which have really made an impact on treatment of  specific cancers in the last few years.  Trastuzumab (Herceptin): A humanized monoclonal antibody against the HER-2 receptor which is now  becoming increasingly important in the treatment of both locally advanced and metastatic breast cancer.    Imatinib mesylate (Gleevec) : An adenosine triphosphate binding selective inhibitor of bcr-abl that has been shown to produce durable complete haematologic and cytogenetic remissions in early  chronic phase CML. It also has remarkable activity  against relapsed and metastatic gastrointestinal stromal tumours (GIST) that characteristically feature a mutation in the c-kit receptor tyrosine kinase gene. 

Figure 4.5 Schematic representation of possible anti-angiogenesis targets, including natural stimulators and inhibitors.

  Ritiximab (Mabthera): The rituximab antibody is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. It is being increasingly  used in combination with chemotherapy to manage many different types of indolent and aggressive B-cell lymphomas.    Bortezomib (Velcade) : Velcade is the first of a new  class of agents called proteasome inhibitors and the first treatment in more than a decade to be approved for patients with multiple myeloma. 

The proteasome is an enzyme complex that exists in all cells and plays an important role in degrading proteins fundamental to all cellular processes, in particular those involved in cell growth and survival. Velcade is a potent but reversible inhibitor of the proteasome. By disrupting normal cellular processes, proteasome inhibition promotes apoptosis. Cancer cells appear to be more susceptible to this effect than normal cells. Due to the reversibility of proteasome inhibition with Velcade, normal cells are more readily able to recover, whereas cancer cells are more likely to undergo apoptosis.

CONCLUSION The majority of conventional chemotherapeutic agents cause cell death by directly inhibiting the synthesis of 

Chapter 4   Mechanisms of anticancer drugs ]  45

DNA or interfering with its function. This means that they are often not tumour-specific and are associated with considerable morbidity. Trials have demonstrated that combination chemotherapy regimens can cause dramatic regression of head and neck tumours, especially when used concomitantly with radiotherapy. Unfortunately, this has not been associated with an increase in survival rates. There is considerable excitement over the development of new target-directed cytotoxic agents. These have been developed to modulate or inhibit specific molecular targets critical to the development of or control of cancer cells. Particular interest has focussed on the field of  monoclonal antibody development, particularly in relation to the epidermal growth factor. Other drugs affecting signal transduction, programmed cell death, transcription regulation, matrix invasion and angiogenesis are currently involved in clinical trials. The results of these are obviously eagerly awaited and will potentially  radically change current therapeutic strategies.

made in relation to more specific targeted therapies. The results of clinical trials with these new agents and their incorporation into management regimens are eagerly awaited. $  The optimum regimen of chemotherapy agents for use in head and neck cancers needs to be defined aiming to improve survival, quality of life and organ function. $   The role of molecular target-specific chemotherapy agents in the management of head and neck cancers needs to become more familiar.

REFERENCES

KEY POINTS 

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Traditional chemotherapy agents interfere with DNA synthesis and function and are classified according to their mechanism of  action. Many agents are associated with significant side-effect profiles. The role of chemotherapy in head and neck  cancer is still being defined, but there is increasing popularity of concurrent chemotherapy and radiotherapy regimens. Current research is focussing on molecular targeted therapy. Recent strategies have looked at the use of  monoclonal antibodies. Drugs are being designed that influence signal transduction, specifically cell cycle regulation, apoptosis, matrix invasion and angiogenesis. Results of clinical trials are eagerly awaited.

Deficiencies in current knowledge and areas for future research The effect of chemotherapy on nonmetastatic head and neck cancers is still being elucidated. The optimum combination of chemotherapeutic agents and the timing of their use in relation to surgery have not been defined, especially in combination with radiotherapy. As this continues to be assessed, significant advances are being

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1. Skipper HE, Schabel FM, Wilcox WS. Experimental evaluation of potential anti-cancer agents. XIII On the criteria and kinetics associated with ‘curability’ of  experimental leukaemia.  Cancer Chemotherapy Reports . 1964;  35 : 1–111. 2. Herscher LL, Cook J. Taxanes as radiosensitizers for head and neck cancer.   Current Opinion in Oncology . 1999;  11: 183–6. 3. Guichard SM, Danks MK. Topoisomerase enzymes as drug targets.  Current Opinion in oncology . 1999;  11: 482–9. 4. Henk JM. Concomitant chemotherapy for head and neck cancer: saving lives or grays?  Clinical Oncology (Royal  College of Radiologists (Great Britain)) . 2001;  13 : 333–5. Brief summary of important issues and trials with respect  to the role of chemotherapy and radiotherapy in head and  neck cancers. 5. Pignon JP, Bourhis J, Domenge C, Designe L. Chemotherapy added to locoregional treatment for head and neck squamous-cell carcinoma: three meta-analyses of updated individual data. Lancet . 2000;  355 : 949–55.   Main meta-  analysis on the effect of chemotherapy on nonmetastatic  head and neck squamous-cell carcinoma. 6. Forastiere A, Koch W, Trotti A, Sidransky D. Head and Neck Cancer. New England Journal of Medicine . 2001; 345: 1890–1900. Very good summary of the important  advances in the treatment of patients with head and  neck cancer and the future importance of molecular  biology. 7. Haffty BG. Concurrent chemoradiation in the treatment of  head and neck cancer.  Hematology/Oncology Clinics of  North America . 1999;  13 : 719–42. 8. Hong WK, Endicott J, Itri LM, Doos W, Batsakis JG, Bell R et al. 13-Cis retinoic acid in the treatment of oral leukoplakia. New England Journal of Medicine . 1986;  315: 1501–5. 9. Hong WK, Lippman SM, Itri LM, Karp DD, Lee JS, Byers RM et al. Prevention of second primary tumours with isotretinoin in squamous-cell carcinoma of the head and neck. New England Journal of Medicine . 1990;  323 : 795–801.

46 ]  PART 1  CELL BIOLOGY  10. Chan G, Boyle JO, Yang EK, Zhang F, Sacks PG, Shah JP et al.  Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck.  Cancer  Research. 1999;  59 : 991–4. 11. Green MC, Murray JL, Hortobagyi GN. Monoclonal antibody therapy for solid tumours. Cancer Treatment  Reviews . 2000;  26 : 269–86. 12. Ke LD, Adler-Storthz K, Clayman GL, Yung AW, Chen Z. Differential expression of epidermal growth factor receptor in human head and neck cancers.  Head and Neck . 1998;  20 : 320–7. 13. Mauizi M, Almadori G, Ferrandina G, Distefano M, Romanini M, Cadoni G et al.  Prognostic significance of  epidermal growth factor receptor in laryngeal squamous cell carcinoma.  British Journal of Cancer . 1996;  74 : 1253–7. 14. Perrotte P, Matsumoto T, Inoue K, Kuniyasu H, Eve BY, Hicklin DJ et al.  Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. Clinical Cancer Research. 1999;  5 : 257–64. 15. Baselga J. The EGFR as a target for anticancer therapy: focus on cetuximab. European Journal of Cancer . 2001; 37: S16–22. 16. Wheeler RH, Spencer S, Buchsbaum D, Robert F. Monoclonal antibodies as potentiators of radiotherapy and chemotherapy in the management of head and neck cancer.  Current Opinion in Oncology . 1999;  11: 187–190. 17. Saleh M, Buchsbaum D, Meredith R, Lalison D, Wheeler R. In vitro  and  in vivo  evaluation of the cytotoxicity of  radiation combined with chimeric monoclonal antibody to the epidermal growth factor receptor. Proceedings for the  American Association for Cancer Research. 1996;  37 : 612 (Abstr. 4197). 18. Herbst RS, Langer CJ. Epidermal growth factor receptors as a target for cancer treatment: the emerging role of IMCC225 in the treatment of lung and head and neck cancers. Seminars in Oncology . 2002;  29 : 27–36. 19. Cohen EE, Rosen F, Stadler WM, Recant WM, Stenson K, Huo D et al. Phase II trial of ZD1839 in recurrent or metastatic squamous cell carcinoma of the head and neck. Journal of Clinical Oncology . May 15, 2003;  21 : 1980–7.

20. Caponigro F. Rationale and clinical validation of epidermal growth factor receptor as a target in the treatment of  head and neck cancer.  Anticancer Drugs . April, 2004;  15 : 311–20. 21. Perez-Soler R. Can rash associated with HER1/EGFR inhibition be used as a marker of treatment outcome? Oncology   (Williston Park, NY). 2003;  17 : 23–8. 22. Folkman J. Seminars in Medicine of Beth Israel Hospital, Boston. Clinical application of research on angiogenesis. New England Journal of Medicine . 1995;  333 : 1757–63. 23. Boehm T, Folkman J, Browder T, O’Reilly MS. Antiangiogenic treatment of experimental cancer does not induce acquired drug resistance.  Nature . 1997;  390: 404–7. 24. Kyzas PA, Stefanou D, Batistatou A, Agnantis NJ. Potential autocrine function of vascular endothelial growth factor in head and neck cancer via vascular endothelial growth factor receptor-2. Modern Pathology . 2005;  18 : 485–94. 25. Hurwitz H, Fehrenbacker L, Novotny W, Cartwright T, Hainsworth J, Heim W et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer.  New England Journal of Medicine . 2004;  350 : 2335–42. 26. Caponigro F, Formato R, Caraglia M, Normanno N, Iaffaioli RV. Monoclonal antibodies targeting epidermal growth factor receptor and vascular endothelial growth factor with a focus on head and neck tumors.  Current Opinion in Oncology . 2005;  17 : 212–7. 27. Caponigro F, Basile M, de Rosa V, Normanno N. New drugs in cancer therapy, National Tumor Institute, Naples, 17–18 June 2004.  Anticancer Drugs . 2005;  16 : 211–21.

FURTHER READING Dancey J, Arbuck S. Cancer Drugs and Cancer Drug Development for the New Millennium. In: Khayat D, Hortobagyi GN (eds). Progress in anti-cancer chemotherapy , Volume IV. 2000: 91–107.  Detailed discussion of the mechanism of novel  chemotherapeutic agents, particularly molecular targeted  therapies.